The intestinal epithelium represents a physical as well as an immunologic barrier, which is in constant contact with approximately 1013-1014 microorganisms. (Gill S R, Pop M, Deboy R T, Eckburg P B, Turnbaugh P J, Samual B S, Gordon J I, Reiman D A, Fraser-Liggett and Nelson K E, 2006. Metagenomic analysis of the human distal gut microbiome. Science 312:1355-1359; Turnbaugh P J, Ley R E, Hamady M, Fraser-Liggett C M, Knight R, and Gordon J I, 2007. The human microbiome project. Nature 449:804-810; Xu J and Gordon J I, 2003. Inaugural Article: Honor thy symbionts. Proc Natl Acad Sci USA 100:10452-10459). Bacteria comprise the vast majority of the intestinal organisms with at least 1000 different species present within the community (Hooper L V, 2009. Do symbiotic bacteria subvert host immunity? Nat Rev Microbiol 7:367-374; Qin J, Li R, Raes J et al. 2010. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464:59-65; Zang T, Breitbart M, Lee W H, Run J Q, Wei C L, Soh S W, Hibberd M L, Liu E T, Rohwer F and Ruan Y, 2006. RNA viral community in human feces: prevalence of plant pathogenic viruses. PLoS Biol 4:e3). Therefore, there is a critical need for mechanisms through which the host is protected from hyper-responsive inflammatory processes due to the presence of an unprecedented amount of antigens while still supporting the growth of commensal bacteria which are beneficial to host health and function.
As a first line of innate immune response, the intestinal epithelium has been found to play an important role in the maintenance and regulation of gastrointestinal homeostasis. For instance, it is currently known that the production of antimicrobial peptides and lectins by enterocytes and Paneth cells (Ayabe T, Satchell D P, Wilson C L, Parks W C, Selsted M E and Ouellette A J, 2000. Secretion of microbicidal alpha-defensins by intestinal Paneth cells in response to bacteria. Nat Immunol 1:113-118; Cash H L, Whitham C V, Behrendt C L and Hooper L V, 2006. Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313:1126-1130; Christa L, Carnot F, Simon M T, Levavasseur F, Stinnakre M G, Lasserre C, Thepot D, Clement B, Devinoy E, and Brechot C, 1996. HIP/PAP is an adhesive protein expressed in hepatocarcinoma, normal Paneth, and pancreatic cells. Am J Physiol 271:G993-1002; Cunliffe R N, Rose F R, Keyte J, Abberley L, Chan W C, and Mahida Y R, 2001. Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease. Gut 48:176-185; Satchell D P, Sheynis T, Shirafuji Y, Kolusheva S, Ouellette A J, and Jelinek R, 2003. Interactions of mouse Paneth cell alpha-defensins and alpha-defensin precursors with membranes. Prosegment inhibition of peptide association with biomimetic membranes. Prosegment inhibition of peptide associations with biomimetic membranes. J Biol Chem 278:13838-13846), the production of mucins by goblet cells (Bergstrom K S, Kissoon-Singh V, Gibson D L, Ma C, Montero M, Sham H P, Ryz N, Huang T, Velcich A, Finlay B B, Chadee K and Valiance B A, 2010. Muc2 protects against lethal infectious colitis by disassociating pathogenic and commensal bacteria from the colonic mucosa. PLoS Pathog 6:e1000902; Van der Sluis M, De Koning B A, et al. 2006. Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection. Gastroenterology 131:117-129) and modulation of epithelial barrier integrity all act in concert to regulate and maintain intestinal immune homeostasis (Rakoff-Nahoum, S., Paglino J, Eslami-Varzaneh F, Edgerg S, and Medzhitov R, 2004. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118:229-241).
Toll-like receptors (TLRs) comprise a family of innate pattern recognition receptors (PRRs) that sense conserved microbial structures known as pathogen-associated molecular patterns (PAMPs) and endogenous danger molecules (Akira S and Takeda K, 2004. Toll-like receptor signaling. Nat Rev Immunol 4:499-511; Medzhitov R, 2007. Recognition of microorganisms and activation of the immune response. Nature 449:819-826; Takeda K, and Akira S, 2007. Toll-like receptors. Curr Protoc Immunol Chapter 14: Unit 14 12). TLR2, a member of this family, recognizes a number of conserved molecular patterns including lipopeptides, lipoteichoic acid and zymosan, through the formation of heterodimers with TLR1 or with TLR6 (Aliprantis A O, Yang R B, Mark M R, Suggett S, Devaux B, Radolf J D, Klimpel G R, Godowski P and Zychlinsky A, 1999. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285:736-739; Brightbill H D, Libraty D H, Krutzik S R, Yang R B, Belisle J T, Bleharski J R, et al. 1999. Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors. Science 285:732-736; Takeda K and Akira S, 2004. TLR signaling pathways. Semin Immunol 16:3-9; Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, and Akira S, 1999. Differential roles of TRL2 and TLR4 in recognition of gramm-negative and gram-positive bacterial cell wall components. Immunity 11:443-451; Takeuchi O, Kawai T, Muhlradt P F, Morr M, Radolf J D, Zychlinsky A, Takeda K and Akira S, 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol 13:933-940; Takeuchi 0, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z, Modlin R L and Akira S, 2002. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol 169:10-14). MyD88 (myeloid differentiation primary response gene 88) and Mal/TIRAP are both required for TLR2 dependent signaling where nuclear factor-kappa B (NF-kB) is activated. While Mal/TIRAP is involved in bridging MyD88 to the TLR2 receptor complex and directing the recruitment of TRAF6 which is necessary for NF-kB activation, Mal binds to the p85α subunit of phosphatidylinositol 3-kinase (PI3K) upon activation of TLR2/TLR6 heterodimer resulting in Akt phosphorylation which consequently leads to macrophage polarization, and cell survival by inhibiting apoptosis. In contrast, TLR2/TLR1 mediated activation of PI3K occurs in the absence of Mal and MyD88 suggesting the presence of another adaptor molecule (Franke T F, Kaplan D R, and Cantley L C, 1997. PI3K:downstream AKTion blocks apoptosis. Cell 88:435-437; Mansell A, Brint E, Gould J A, O'Neill La and Hertzog P J, 2004. Mal interacts with tumor necrosis factor receptor-associated factor (TRAF)-6 to mediate NF-kappaB activation by toll-like receptor (TLR)-2 and TLR4. J Biol Chem 279:37227-37230; Santos-Wierra S et al., 2009. Mal connects TLR2 to PI3 Kinase activation and phagocyte polarization. EMBO J 28:2018-2027). Activation of PI3K pathway as a downstream effect of TLR2 activation has also been shown to augment the tight junction-associated epithelial barrier integrity possibly by acting as a surveillance receptor, which monitors luminal bacteria and translocation of pathogens (Cario E, Gerken G and Podolsky D K, 2007. Toll-like receptor 2 controls mucosal inflammation by regulating epithelial barrier function. Gastroenterology 132:1359-1374; Cario E, Gerken G and Podolsky D K, 2004. Toll-like receptor 2 enhances ZO-1 associated intestinal epithelial barrier integrity via protein kinase C. Gastroenterology 127:224-238; Podolsky D K, Gerken G, Eyking A and Cario E, 2009. Colitis-associated variant of TLR2 causes impaired mucosal repair because of TFF3 deficiency. Gastroenterology 137:209-220).
Amyloids
Amyloids that possess a fibrillar cross-β sheet quaternary structure are produced both by humans and bacteria. While amyloids in humans are mostly associated with complex diseases, functional amyloids that serve a role in physiological processes such as melanin production and blood clotting have been reported (Aigelsreiter A et al. 2007. How a cell deals with abnormal proteins. Pathogenic mechanisms in protein aggregation diseases. Pathobiology 74:145-158; Brandan E and Inestrosa N C, 1993. Extracellular matrix components and amyloid in neuritic plaques of Alzheimer's disease. Gen Pharmacol 24:1063-1068; Hull R L et al., 2004. Islet amyloid: a critical entity in the pathogenesis of type 2 diabetes. J Clin Endocrinol Metab 89:3629-3643; Leonhardt R M et al. Endoplasmic reticulum export, subcellular distribution, and fibril formation by Pmel17 require an intact N-terminal domain junction. J Biol Chem 285:16166-16183; Pfefferkorn C M, McGlinchey R P and Lee J C, 2010. Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17. Proc Natl Acad Sci USA 107:21447-21452; Theos et al., 2005. The Silver locus product Pmel17/gp100/Silv/ME20: controversial in name and in function. Pigment Cell Res 18:322-336).
In bacteria, amyloids function as a component of the extracellular matrix in biofilms of commensal organisms such as spore forming Bacillus subtilis, Pseudomonas fluorescens or human pathogens such as Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Citrobacter freundii, Enterobacter sakazakii and Escherichia coli (Alteri et al., 2007. Mycobacterium tuberculosis produces pili during human infection. Proc Natl Acad Sci USA 104:5145-5150; Blanco L P et al., 2011. Diversity, biogenesis and function of microbial amyloids. Trends Microbiol 20:66-73; Chapman M R et al. 2002. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 295:851-855; Collinson S K et al, 1996. Salmonella enteritidis agfBAC operon encoding thin, aggregative fimbriae. J Bacteriol 178:662-667; Dueholm M S, et al. 2010. Functional amyloid in Pseudomonas. Mol. Microbiol 77(4):1009-1020; Larsen P et al., 2007 Amyloid adhesins are abundant in natural biofilms. Environ Microbiol 9:3077-3090; Romero C et al, 2010. Amyloid fibers provide structural integrity to Bacilus subtilis biofilms. Proc Natl Acad Sci USA 107:2230-2234; Zogaj X et al. 2003. Production of cellulose and curli fimbriae by members of the family Enterobacteriaceae isolated from the human gastrointestinal tract. Infect Immun 71:4151-4158).
Curli fibrils produced by enteric bacteria including Salmonella spp and E. coli are the best-characterized bacterial amyloid to date. Earlier studies have shown that curli fibrils activate the immune system inducing the production of inflammatory cytokines in a mouse model of sepsis as well as urinary tract infection induced by E. coli (Bian Z, Brauner A, Li Y and Normark S, 2000. Expression of and cytokine activation by Escherichia coli curli fibers in human sepsis. J Infect Dis 181:602-612; Bian Z, Yan Z Q, Hansson G K, Thoren P and Normark S, 2001. Activation of inducible nitric oxide synthase/nitric oxide by curli fibers leads to a fall in blood pressure during systemic Escherichia coli infection in mice. J Infect Dis 183:612-619; Kai-Larsen Y et al. 2010. Uropathogenic Escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide LL-37. PLoS Pathog 6:e1001010; Tukel C et al, 2010. Toll-like receptors 1 and 2 cooperatively mediate immune responses to curli, a common amyloid from enterobacterial biofilms. Cell Microbiol 12:1495-1505; Tukel C et al., 2005. CsgA is a pathogen-associated molecular pattern of Salmonella enterica serotype Typhimurium that is recognized by Toll-like receptor 2. Mol Microbiol 58:289-304; Tukel C et al. 2009. Responses to amyloids of microbial and host origin are mediated through Toll-like receptor 2. Cell Host and Microbe 6(1):45-53).
Curli fibrils are indeed a pathogen-associated molecular pattern (PAMP) that is recognized by the TLR2/1 heterodimer. Interestingly, TLR2 responds not only to curli fibrils but also recognizes host amyloids such as β-amyloid 1-40 and β-amyloid 1-42 of Alzheimer's plaques as well as serum amyloid A, an acute phase protein (Cheng et al., 2008. Cutting edge: TLR2 is a functional receptor for acute-phase serum amyloid A. J Immunol 181:22-26; He R L et al. 2009. Serum amyloid A induces a G-CSF expression and neutrophilia via Toll-like receptor 2. Blood 113:429-437; Jana M et al. 2008. Fibrillar amyloid-beta peptides activate microglia via TLR2: implications for Alzheimer's disease. J Immunol 181:7254-7262; Reed-Geaghan E G et al. 2009CD14 and toll-like receptors 2 and 4 are required for fibrillar a{beta}-stimulated microglial activation. J Neurosci 29:11982-11992; Tukel C et al, 2010. Toll-like receptors 1 and 2 cooperatively mediate immune responses to curli, a common amyloid from enterobacterial biofilms. Cell Microbiol 12:1495-1505; Udan M L et al. 2008. Toll-like receptors 2 and 4 mediate Abeta(1-42) activation of the innate immune response in a human monocytic cell line. J Neurochem. 104(2):524-533). In fact, TLR2 recognizes the conserved quaternary β-sheet structure that is common to amyloids of all distinct origins.
Amyloids have also been reported to be present in the biofilms of members of Bacteriodetes and Firmicutes, the predominant phyla found in the gastrointestinal tract (Larsen P et al., 2007. Amyloid adhesins are abundant in natural biofilms. Environ Microbiol 9:3077-3090; Lay C et al., 2005. Design and validation of 16S rRNA probes to enumerate members of the Clostridium leptum subgroup in human faecal microbiota. Environ Microbiol 7:933-946).
Frequently, inflammatory disorders or diseases occur at or near an epithelium, such as that of the skin, the cornea or the gastrointestinal lining. Inflammatory disorders or diseases of the epithelium include the following.
Inflammatory Bowel Disease
Crohn's disease (CD) and ulcerative colitis (UC), and to a lesser extent, indeterminate colitis and infectious colitis, are collectively referred to as inflammatory bowel disease (IBD). Inflammatory bowel diseases are chronic recurrent inflammatory diseases of unclear etiology, affecting the small intestine and colon. IBD can involve either or both the small and large bowel. These disorders or diseases fall into the category of “idiopathic” IBD because the etiology for them is unknown.
Pathologic findings are generally not specific, although they may suggest a particular form of IBD. “Active” IBD is characterized by acute inflammation. “Chronic” IBD is characterized by architectural changes of crypt distortion and scarring. The term “crypt” refers to a deep pit that protrudes down into the connective tissue surrounding the small intestine. Crypt abscesses (active IBD characterized by the presence of neutrophils in crypt lumens) can occur in many forms of IBD, not just UC. Under normal conditions the epithelium at the base of the crypt is the site of stem cell proliferation and the differentiated cells move upwards and are shed 3-5 days later at the tips of the villi. This normal process, necessary for proper bowel function, is interrupted by IBD.
UC involves the colon as a diffuse mucosal disease with distal predominance. The rectum is virtually always involved, and additional portions of colon may be involved extending proximally from the rectum in a continuous pattern. Most often the UC occurs in young people 15 to 40 years of age. UC occurs only in the inner lining of the colon (large intestine) or rectum. When it is localized in the rectum, it is called “proctitis.”
CD is a chronic inflammatory disease that has periods of remission (time when a person feels well) and relapse (when a person feels ill). CD is an inflammation and ulceration process that occurs in the deep layers of the intestinal wall. The most common areas affected are the lower part of the small intestine, called the ileum, and the first part of the colon. This type of CD is called ileocolitis. CD can infrequently affect any part of the upper gastrointestinal tract. Aphthous ulcers, which are similar to cold sores, are common. Ulcers can also occur in the esophagus, stomach and duodenum.
Therapy for IBD has historically included administration of corticosteroids. However, drawbacks of long term corticosteroid therapy include masking (or induction) of intestinal perforation, osteonecrosis and metabolic bone disease. Additional problems relate to development of corticosteroid dependency (Habnauer, New England Journal of Medicine, 334(13):841-848). Aminosalicylates such as sulfasalazine and mesalamine have been used to treat mild or moderately active UC and CD, and to maintain remission (Id at 843). Immunomodulatory drugs such as azathioprine and mercapto purine have been used in long term treatment for patients with IBD. Common complications with both of these drugs include pancreatitis, which occurs with an incidence of 3-15% of patients, and bone marrow suppression, which requires regular monitoring. More potent immunosuppressive drugs such as cyclosporine and methotrexate have been employed, but toxicity of these drugs limits their use to specific situations of refractory disease states. Other therapeutic approaches include antibiotic therapy and nutritional therapy. Often, therapy involves a combination of the above-described drug therapies in addition to surgical resection of the bowel.
There is no cure for IBD. Ultimately, the chronic and progressive nature of IBD demands a long-term treatment that maximizes the local anti-inflammatory effect while minimizing the global systemic effect on the immune system.
Chronic inflammatory disorders or diseases such as CD typically demonstrate periods of remission between intervals when the inflammatory is active and requires acute treatment. This is an example of a circumstance wherein it is known beforehand that an individual will develop, or is likely to develop an inflammatory disorder or disease.
Inflammatory Skin Disorders or Diseases
1. Psoriasis
Another chronic inflammatory condition of the epithelium is psoriasis. Psoriasis is a chronic, recurrent, papulosquamous plaque on areas of trauma such as the elbow, knee or scalp, though it may appear elsewhere on the skin. Psoriasis may coexist with lupus erythematosis in some individuals. Current treatments include topical administration of psoralens. “Psoralens” refers to a group of substances found in many different plants; especially psoralea corylifolia. Psoralens interact with nucleic acids and are also sued as research tools. Psoriasis is also treated by long-wave ultraviolet radiation. Neither treatment cures or prevents recurrence of psoriasis symptoms.
2. Atopic Dermatitis/Eczema
Atopic dermatitis is a chronic disease that affects the skin. In atopic dermatitis, the skin becomes extremely itchy. Scratching leads to redness, swelling, cracking, “weeping” clear fluid, and finally, crusting and scaling. In most cases, there are periods of exacerbations followed by periods of remissions. Although it is difficult to identify exactly how many people are affected by atopic dermatitis, an estimated 20% of infants and young children experience symptoms of the disease. Approximately 60% of these infants continue to have one or more symptoms of atopic dermatitis in adulthood. Thus, more than 15 million people in the United States have symptoms of the disease.
3. Contact Dermatitis
Contact dermatitis is a reaction that occurs when the skin comes into contact with an allergen, i.e., a substance to which the body is allergic. Allergens, though harmless to most individuals, cause an allergic reaction in individuals having a congenital or acquired hypersensitivity to the specific allergen.
Aphthous Ulcers (Oral)
Although the cause of aphthous ulcers remains unknown, many physicians believe they are caused by autoimmune phenomena, which cause the destruction of discrete areas of the oral mucosa which leads to oral ulceration. Among the cytokines present in these active areas of ulceration, TNF-α appears to play a predominant role.
Peptic Ulcer Disease
Inhibition of gastric acid secretion with H2-receptor antagonists and, more recently, blockers of H+, K+-ATPase (also known as the proton pump) has been the mainstay of therapy for peptic ulcer disease. The pathophysiology of peptic ulcers remains obscure. An appreciation of the complexity of the physiology of the gastric mucosa has led to a hypothesis that peptic ulcers are the result of an imbalance in the relative importance of aggressive (acid, pepsin) and protective (mucus, bicarbonate, blood flow, prostaglandins, etc.) factors. Infection of the mucosa of the human gastric antrum with the bacterium Helicobacter pylori has been widely accepted as the cause of chronic, active, type B gastritis. Further, this form of gastritis has been linked directly to peptic ulcer disease by studies showing that eradication of H. pylori reverses this gastritis and prevents duodenal ulcer relapse.
There remains a need for a method of treating or preventing inflammatory disorders or diseases of the epithelium.